Time-resolved fluorescence microscopy combined to Scanning ElectroChemical Microscopy (SECM): a new way to visualize photo-induced electron transfer quenching with an electrofluorochromic probe

The combination of Scanning Electrochemical Microscopy (SECM) and Time-Resolved Fluorescence Microscopy was successfully implemented to investigate the control of the fluorescence of a tetrazine derivative in solution according to its redox state, a phenomenon called electrofluorochromism. The normalized fluorescence intensity difference between the 'on' and 'off' states can be tuned by the tip potential and the distance between the polarized tip and the substrate. It is demonstrated for the first time that luminescence lifetime can be modulated as well, an evidence that a quenching process contributes to the fluorescence intensity drop. The experimental results demonstrate that this quenching is due to the electron transfer reaction between the excited fluorophore and the electrogenerated anion radical. A mixed dynamic and static quenching mechanism is considered to fit the variations of both luminescence intensity and lifetime ratios vs. the coulombic charge. The pertinence of a static quenching is validated by DFT calculations. Insulating (glass) and conductive (ITO) substrates have been tested and optical approach curves based on fluorescence lifetime modulation have been obtained in each case that are more sensitive than the electrochemical one based on the tip current. Finally, numerical simulations have been performed to fit these optical approach curves and validate the role of the electron transfer quenching in the variation of fluorescence intensity and lifetime.